Stem cell therapy in diabetic foot patients: where are we now?

154 Kirana et al.

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Stem cell therapy in diabetic foot patients: where are we now?
Stanley Kirana, Diethelm Tschöpe, Bernd Stratmann
Diabetes Center, Heart and Diabetes Center NRW,University Clinic of Ruhr University Bochum; Bad Oeynhausen, Germany

Abstrak
Kaki Diabetik (KD) merupakan penyakit penyerta Diabetes Mellitus (DM). DM merupakan salah satu penyebab utama
kasus amputasi non-traumatik di Jerman, dengan penyakit arterial perifer berat (PAP) dengan iskemia kritis tungkai yang
menjadi masalah utama. Meskipun teknik modern tersedia, intervensi perkutan dan pembedahan pemulih vaskularisasi masih
terbatas. Masalah ini menyebabkan peningkatan jumlah amputasi pada pasien dengan diabetes mellitus. Proses fisiologik
angiogenesis, vaskulogenesis dan arteriogenesis mengarahkan ke pertumbuhan pembuluh darah kolateral pada keadaan
penyakit penyumbatan pembuluh arterial penyebab iskemia tungkai. Pada praktik klinik respons angiogenik endogenik
seringkali terganggu. Angiogenesis terapetik merupakan penerapan bioteknologi untuk merangsang pembentukkan
pembuluh darah baru via (melalui) aplikasi lokal faktor penumbuh pro-angiogenik dalam bentuk protein rekombinan, atau
terapi gen; atau dengan implantasi sel progenitor atau sel punca yang akan mensintesa sitokin angiogenik multipel. Artikel
review ini merangkum fungsi endotelial dan disfungsi pada DM, mekanisme homing, metode transplantasi dan status uji
klinik di bidang sel punca untuk pengobatan iskemia tungkai. (Med J Indones 2011; 20:154-60)

Abstract
Diabetic foot (DF) occurs as a concomitant illness of diabetes mellitus (DM). DM is one of the main causes of nontraumatic amputation in Germany with severe peripheral arterial disease (PAD) with critical limb ischemia (CLI) being of
major concern. Although modern techniques are available surgical vascularisation and percutaneous intervention are limited.
This problem leads increasing numbers of limb amputations in patients with diabetes mellitus. Thephysiological process
of angiogenesis, vasculogenes is and arteriogenesis contribute to the growth of collateral vessels in response to obstructive
arterial disease causing limb ischemi. In clinical practice the endogenous angiogenic response is often impaired. Therapeutic
angiogenesis is an application of biotechnology to stimulate new vessel formation via local administration of pro-angiogenic
growth factors in the form of recombinant protein, or gene therapy,or by implantation of progenitor cells or stem cells that
will synthe size multiple angiogenic cytokines. This review summarises the endothelial function and dysfunctionin DM, the
mechanism of homing, the transplantation method and the status of clinical trials in stem cell field to treat limb ischemia.
(Med J Indones 2011; 20:154-60)
Key words: diabetes mellitus, endothelial progenitor cells, peripheral arterial disease, stem cells, therapeutic angiogenesis

Diabetes mellitus (DM) is known as an emerging chronic disease world wide. DM is accepted as cardiovascular
risk factor and therefore plays an important role in the
pathogenesis of cardiovascular disease. Peripheral artery disease (PAD) is one of the major health problems
resulting from macrovascular complication in diabetic
patients. As a manifestation of atherosclerosis, PAD is
characterized by atherosclerotic occlusive disease of the
lower extremities and is a marker for atherothrombotic

disease in other vascular beds. PAD is present in approximately one-half of all patients with foot ulcers.1-4 Along
with polyneuropathy, PAD causes foot ulceration which
often leads to lower limb amputations.
DM causes almost 50% of all non traumatic lowerextremity amputations world wide. It is estimated that
the life time risk for amputation in diabetic patients is
10–15%, which is 10-30 times higher in comparison
to the general population.5-8 In Germany the number
of limb amputation is increasing. According to the newest data from German Association of Angiology, which
were released during the Scientific Meeting in Mannheim
in September 2008, about 60.000 amputations are performed every year. Compared to european countries such
Correspondence email to: dtschoepe@hdz-nrw.de

as Denmark and The Netherlands, Germany is leader in
the number of limb amputation. The main factors inducing these amputations are diabetic foot (DF) or arteriosclerosis. Almost every 3rd patient suffers from diabetes
mellitus. An amputation for elderly patients resembles a
fatal step in their quality of life.
In the other hand, the patients are often late to seek specialized professional care or they are not treated in a vascular
center. These factors along with pathology-anatomical
factors limit the possibilities for revascularisation.
The strengths and limitations of surgical revascularisation in PAD are well known. Surgical revascularisation

and percutaneous interventions are limited to patients
with critical limb ischemia (CLI) and those with disabling claudication (Fontaine Stadium IIb–IV) due to
discrete proximal disease.1,9
Endothelial functions and dysfunctions
As key elements in the maintenance of tissue homeostasis, blood vessels serve as the conduits of circulation,
transporting nutrients and oxygen to organs and tissues,
and removing destructive catabolites and xenobiotics
from the blood flow.10

Vol. 20, No. 2, May 2011

For a long time two specialized endothelial functions
were accepted: gas exchange in pulmonary circulation
and fenestration in hepatic and splenic circulation. Under normal homeostatic conditions, the endothelium
resists vasospasm, prevents leukocyte and platelet adhesion to the vessel wall, favours fibrinolysis, combats
coagulation of blood, and inhibits the proliferation of
vasculars mooth muscle cells.
During the last two decades, accumulating evidence
has described the vascular endothelium as an active endocrine, paracrine, and autocrine organ, indispensable
for the maintenance of vascular homeostasis. Altered

homeostasis induced by various stimuli may cause localized alterations, or ‘endothelial dysfunction’, of the
antihemostatic properties, vascular tone, heightened
leukocyte adhesion, and increased production of cytokines and growth factors. The dramatic change of endothelial interactions with blood leukocyte soccurring
in inflammation provides an example of endothelial
activation.10,12,13

Stem cell therapy of diabetic foot 155

both.21,22 Therapeutic angiogenesis has been studied
many times for the treatment of patients with peripheral arterial occlusive disease who do not qualify for
surgical revascularisation or radiologic intervention.
Angiogenesis can be achieved by introducing growth
factors as mature proteins or as complementary DNA
carrying vector systems (cDNA-plasmid) or stem cells
which contain endothelial progenitor cells (EPCs) and
mesenchymal cells.23-27
The following sections discuss various methods of using stem cell therapies to induce angiogenesis in DF.

Diabetes mellitus and endothelial dysfunction
Vascular diseases, including atherosclerosis, medial

calcification, and microangiopathy, are prevalent in patients with DM and are the principal causes of morbidity and mortality in these individuals. Atherosclerosis
occurs earlier in patients with diabetes, frequently with
greater severity and more diffuse distribution.
Hyperglycemia per se causes endothelial dysfunction. Healthy humans exposed to a hyperglycaemic
clamp sufficient to increase forearm glucose concentration experience impaired endothelium-dependent
vasodilation.10,14,15 Hyperglycemia also decreases flowmediated endothelium-dependent vasodilation of the
brachial artery of healthy subjects.14,16,17 Hyperglycemia may decrease the bioavailability of nitric oxide
(NO) through multiple mechanisms (Figure1). Additionally, hyperglycemia may increase the formation
of oxygen-derived free radicals that inactivate NO or
cause intracellular signalling disturbances that inhibit
nitric oxide synthase (NOS) activity and thereby reduce NO production. Hyperglycemia is also associated
with increased oxidative stress. Increased inactivation
of NO by oxygen-derived free radicals and decreased
production of NO by NOS reduce NO levels in the vascular milieu.14,17-20
Rationale for stem cell therapy in diabetic foot
The essential part of normal wound healing is the formation of new blood vessels with in the provisional
wound matrix that is referred to as granulation tissue.
Neovascularisation of the granulation tissue occurs by
the processes of angiogenesis or vasculogenesis, or

Figure 1. Sources for embryonic and adult stem cells28

Definition of stem cells
All life forms begin with a stem cell, which is defined
as a cell that has the dual ability to self-renew and to
produce progenitors and different types of specialized
cells in the organism.28
Regardless of their sources, stem cells are defined by
their indefinite capability of self-renewal (proliferation) and unlimited potential to generate specialized
tissue cells (differentiation) (Figure2). Currently, scientists and clinical researchers are working on two major
types of stem cells, embryonic and adult, which share
three characteristic properties: 1. Stem cells are premature, undifferentiated, or unspecialized. However,
unspecialized stem cells can generate specialized cells,
including heart muscle, blood vessels, blood cells, or
nerve cells. 2. Stem cells can divide and renew themselves for long periods. Unlike other cells–which normally do not replicate by themselves–stem cells may
replicate, or proliferate, many times. 3. Stem cells can
respond to exogenous or endogenous signals by generating specialized cell types. When unspecialized stem
cells develop into specialized cell, the process is called
differentiation.29

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Med J Indones

Figure 2. Isolation, cultivation and genetic engineering of endothelial progenitor cells (EPCs) for therapeutic application. EPC scan be isolated from the BM-MNC, peripheral blood or umbilical chord blood with or
without further selection and purification.The mononuclear cells are expanded ex vivo under endothelialspecific growth conditions and may be genetically modified to over express one or several therapeutic
genes.The differentiated cells are then used in transplantation protocols for rescue and repair of damaged
tissue such as infarcted myocardium, ischemic limb or injured muscle. The cells may also be used for endothelialisation of damaged blood vessels and vascular prosthetic grafts and in tissue engineering. (Melo
LG, Pachori AS, Kong D et al. Endothelium-targeted gene and cell-based therapy for cardiovascular disease. In: De Caterina R, Libby P, eds. Endothelial Dysfunctions and Vascular Disease. Oxford: Blackwell
Futura; 2007:385)

Adult and embryonic stem cells
Human embryonic stem cells (ESCs) used for research
have been extracted from embryos created by in vitro
fertilization done by the group of James Thomson in
1998 and they reported on the etablishment of human
ESC lines. They concluded that the ESCs have potential to form most cell types of the adult body over almost unlimited periods.28,30-32
The adult body has a small number of adult or somatic
stem cells in some tissues and organs. Such adult stem
cells (ASCs) have been known to posses the ability to

regenerate the corresponding tissue from which they
are derived. Hematopoietic stem cells (HSCs), for example, continuously regenerate the circulating blood
cells and cells of the immune system during the life
span of the organism. Based on animal studies, many
researchers have recently claimed that ASCs might
exhibit developmental potentials comparable to those
exhibited by ESCs. ASCs have the ability to regenerate the tissue from which they are derived over the life
span of the individual, while ESCs have the potential to

form most cell types of the adult body over very long periods of in vitro cultivation.28,29,32-38 However, until now the
use of human ESCs for research is prohibited in Germany.
Many clinical research centers in Germany are focusing to
develop the potential of ASCs.
Endothelial progenitor cells for cardiovascular regeneration
The differentiation of mesodermal cells to angioblasts
and subsequent endothelial differentiation was believed
to occur exclusively in embryonic development. Asahara et al. reported that purified CD34+ hematopoietic
progenitor cells from adults can differentiate ex vivo to
an endothelial phenotype.40,41 These cells were named
“endothelial progenitor cells” (EPC). Shi et al. reported in 1998 the existence of ‘circulating bone marrowderived endothelial progenitor cells’ (CEPC).58 This is

actually a similar finding to Asahara. EPC or CEPC were
defined as cells, which express both, hematopoietic stem
cell markers such as CD34 and an endothelial marker
protein such as the VEGF-receptor KDR.

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CD34 is not expressed exclusively on hematopoietic
stem cells (HSC) but also on mature endothelial cells.
Further studies used the more immature HSC marker
CD133 also known as prominin or AC 133. Purified isolated CD 133+ cells also differentiated to endothelial cells
in vitro.41,42 However, the biological function of CD133
remains unclear. It is not well known whether CD133
only represents a surface marker or has a functional activity involved in regulation of neovascularisation.41
The characterization of EPC becomes particularly difficult when cells are expanded and cultured ex vivo, since
the culture conditions (culture supplements such as fetal calf serum and cytokines or different plastic types)
rapidly change the phenotype of the cells. Moreover,
continuous cultivation was shown to increase endothelial differentiation, as evidenced by elevated endothelial marker protein expression.

Smooth muscle cells (SMC) and endothelium are two
essential structures for the architecture of the blood
vessels. Vascular stem cells, a group of multipotent heterogenous stem cells, largely fall into two subgroups:
endothelial and SMC progenitors. EPC develop closely
from CD34+ to bone marrow stem cells, in particular those committed to the monomyeloid cell lineage,
whereas SMC progenitors may belong or originate together in mesenchymal stem cells (MSC), which are
negative for CD34. Atherosclerosis causes arterial wall
damage and impairs the capacity of both vascular stem
cell types to regenerate neovascular tissue, or may trigger abnormal proliferation or death by apoptosis under
different pathological conditions.29
Mechanism of vessel formation
Angiogenesis is defined as the sprouting of new capillaries from existing vascular structure, a process that is
triggered by endothelial cell migration and proliferation. Remodelling of the extra cellular matrix (ECM),
tubule formation and expansion of the surrounding vascular tissue are key elements of angiogenesis.
The in situ formation of new blood vessels from ECP
which differentiated into endothelial cells and fuse into
luminal structures is called vasculogenesis.
Meanwhile, arteriogenesis refers to an increase in the
calibre of pre-existing arteriolar collateral connections
by recruitment of perivascular cells and expansion and

remodelling of the extracellular matrix. Arteriogenesis
increases the size and wall thickness of collateral vessels, and shear stress (rather than hypoxia) seems to be
the main factor that stimulates arteriogenesis.9
The key steps in vessel formation–namely endothelial
cell activation, migration, proliferation and reorgani-

sation– are tightly regulated in a complex balance between pro-and anti-angiogenic mechanisms (Table1).9
Table 1. Factors that stimulate and inhibit angiogenesis
Stimulators

Inhibitors

VEGF
aFGF
bFGF
PDGF
TGFα/ß
TNFα
HGF
Angiogenin
PIGF
Angiopoietin
Oestrogen

Angiostatin
Thrombospondin
Endostatin
Troponin-I
TIMPs
Suramin

Nitric oxide

VEGF: vascular endothelial growth factor; aFGF, bFGF: acid and basic fibroblast
growth factor (FGF-1 and-2, respectively); PDGF: platelet-derived growth factor;
TGF: transforming growth factor; TNF: tumour necrosis factor; HGF: hepatocyte
growth factor; PIGF: placental growth factor; TIMPs: tissue inhibitors of metalloproteinases.

The most important pro-angiogenic factors are vascular
endothelial growth factor (VEGF) and basic fibroblast
growth factor (bFGF), also known as FGF-2. VEGF is
an endothelial cell specific mitogen that is markedly upregulated by hypoxia, and plays an important role in endothelial cell proliferation, differentiation and survival.
FGFs are non-secreted growth factors that are released
only during cell death or ischemic cell injury. Clinical
and biochemical factors also influence the formation of,
and biological response to different angiogenic growth
factors. Hypoxia, for example, is the most potent inducer of angiogenesis,44 principally via up-regulation
of VEGF, whereas diabetes mellitus and increased levels of cholesterol and lipoprotein(a) are associated with
a reduced angiogenic response.9
As bone marrow-derived stem and progenitor cells
home to sites of ischemia, this may allow the local release of factors acting in paracrine manner on the surrounding ischemic tissue. Bone marrow-derived mononuclear cells release angiogenic growth factors such
as VEGF, bFGF and angiopoietins, thereby enhancing
the local angiogenic response.9,41,44 This mechanism is
named ‘homing’ and it is the most likely theory which
represents the reality in ischemic process.
During tissue injury, e.g. wound, the formation of new
blood vessels is needed. Neovascularization of the
wound’s granulation tissue occurs by the processes
mentioned above. However, in diabetic patients these
angiogenic mechanisms are reduced.

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The idea to induce neovascularisation in the wound area
or ischemic area is now a hot topic in regenerative medicine. There are a lot of clinical trials in this area, however
only few centers world wide perform these studies.
Stem cell transplantation
Until now, there are a lot of protocols, hypotheses, debates and discussions about the best methods of stem cell
transplantation. Most clinical studies are performing 2
methods of stem cell transplantation: intra-arterial and
intra-muscular.
The concept of intra-arterial stem cell transplantation
assures that the stem cells reach all stenosis sites antegradely. We believe that the stenosis area is the ischemic area which stimulates the homing effect of the

cells.The intra-muscular application is based on the
ischemic musculature which could be supplied with a
high concentration of stem cells through several local
depots. Whether the cells reached the ischemic area by
blood flow remains unclear. In theory, it induces the
homing effect and then stimulates the angiogenesis or
vasculogenesis in the ischemic area.45-47
Current clinical trial
There are a lot of clinical trials running on this topic
(Table 2). However, the outcomes have not been published yet. Only some studies published their results
or case reports.25,45,46 A multi center study has not been
performed to take conclusion of stem cell therapy. To
what we know, the data from other centers as well as
ours show positive results (Figure 3).

Table 2. List of stem cell clinical trials in critical limb ischemia as listed in Clinicaltrial.gov from September 2007
Location
USA
USA
USA
USA

Status
Recruiting
Recruiting
Recruiting
Recruiting

USA
USA
Japan
Japan
Korea

Recruiting
Recruiting
Recruiting
Recruiting
Recruiting

The Netherlands
The Netherlands
Italy
France
Denmark
Germany
Germany

Recruiting
Recruiting
Recruiting
Recruiting
Recruiting
Finished
Recruiting

Trial name
ALDHbr Cells for Critical Limb Ischemia
Safety Study of Using Stem Cells to Stimulate Development of New Blood Vessels in Peripheral Vascular Disease
Combination Stem Cell Therapy for the Treatment of Severe Leg Ischemia
Vascular Repair Cells (VRC) Treatment of Patients With Peripheral Arterial Disease Related Critical Limb Ischemia
(RESTORE-CLI)
Study of Autologous Bone Marrow Concentrate for theTreatment of CLI
StemCells for Treating Critical Ischemia
Stem Cell Study for Patients With Leg Ulcer / Gangrene
TACT-NAGOYA: Therapeutic Angiogenesis Using Cell Transplantation
Study for Safety and Efficiency of Therapeutic Angiogenesis for Patients With Limb Ischemia by Transplantation of
Human Cord Blood Mononuclear Cell
Intra-Arterial Stem Cell Therapy for Patients With Chronic Limb Ischemia (CLI)
Autologous Bone Marrow-Derived Mononuclear Cells for Therapeutic Arteriogenesis in Patients With Limb Ischemia
AutologousBone Marrow Cell Treatment in Peripheral Atherosclerosis
Cell Therapy in Chronic Limb Ischemia
Treatment of Severe Limb Ischemia With Autologous Bone Marrow Derived Mononuclear Cells
Novel Therapy of PAD by Combined Transplantation of BMCs (TAM-PAD)
Autologous Bone Marrow Transplantation for Critical Limb-Threatening Ischemia

Figure 3. Bone marrow stem cell therapy in diabetic foot (Bad Oeynhausen Working Group, Germany). This patient had a chronic limb
ischemia with forefoot necrosis. Distal stenosis A. tibialis anterior and A. dorsalis pedis without collateralisation was verified
by angiography. Discussion between vascular surgeon, intervention angiologist and internist revealed no surgical or interventional therapeutic option. Due to the stenosis location and vascular structure, no possibilities to perform bypass or other
intervention were seen. The patient agreed with the autologous bone marrow stem cell therapy.
Left picture shows the wound status before intra muscular stem cell transplantation. Right picture shows the complete wound healing 20
weeks after stem cell transplantation. Small calibre new vessel formation was proved by angiography. (With permission from Kirana,
Stratmann et al. Wound therapy with autologous bone marrow stem cells in diabetic patients with ischaemia- induced tissue ulcers affecting
the lower limbs. Int J Clin Pract 2007;61:690-2)

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Vol. 20, No. 2, May 2011

In conclusion, bone marrow derived EPCs are the newest cellular target that may be used to influence postnatal neovascularisation. The formation of new blood
vessels, including collaterals, is a complex physiological process that occurs in adults in response to tissue
injury or ischemia.
The stem cell transplantation to induce vessel formation is promising and could be a new therapeutic option
in the future to treat limb ischemia without options of
revascularization. However, there is still much to learn
about the optimum treatment modality, dosing frequency and route of administration, especially in patients
with diabetes mellitus. We know that these patients
have a reduced capability of vascular self-renewing.
Until now, there are only some reports and clinical trials which have already been finished with the whole
evaluations and they show an improvement of blood
perfusion in ischemic area. Moreover, multicenter studies with large numbers of patients are expected to give
more information about stem cell therapy.
Acknowledgments
The Bad Oeynhausen stem cell trial was supported by
Aastrom Bioscience Inc., Ann Arbor–USA. We thank
Dr. E. Danch of the Vascular Surgery, Bad Oeynhausen
General Hospital, Germany for his critical opinion of
patient recruitment. We also thank all colleagues in Diabetes Center NRW, Bad Oeynhausen for helping us in
patient recruitment and performing diagnostic.

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